First report of Planomicrobium okeanokoites associated with Himantothallus grandifolius (Desmarestiales, Phaeophyta) from Southern Hemisphere

Gram-positive, aerobic, motile, rod-shaped, mesophilic epiphytic bacterium Planomicrobium okeanokoites was isolated from the surface of endemic species Himantothallus grandifolius in Larsemann Hills, Eastern Antarctica. The diversity of epiphytic bacterial communities living on marine algae remains primarily unexplored; virtually no reports from Antarctic seaweeds. The present study used morpho-molecular approaches for the macroalgae and epiphytic bacterium characterization. Phylogenetic analysis was performed using mitochondrial genome encoded COX1 gene; chloroplast genome encodes rbcL; nuclear genome encoded large subunit ribosomal RNA gene (LSU rRNA) for Himantothallus grandifolius and ribosomal encoded 16S rRNA for Planomicrobium okeanokoites. Morphological and molecular data revealed that the isolate is identified as Himantothallus grandifolius, which belongs to Family Desmarestiaceae of Order Desmarestiales in Class Phaeophyceae showing 99.8% similarity to the sequences of Himantothallus grandifolius, from King George Island, Antarctica (HE866853). The isolated bacterial strain was identified on the basis of chemotaxonomic, morpho-phylogenetic, and biochemical assays. A phylogenetic study based on 16S rRNA gene sequences revealed that the epiphytic bacterial strain SLA-357 was closest related to the Planomicrobium okeanokoites showing 98.7% sequence similarity. The study revealed the first report of this species from the Southern Hemisphere to date. Also, there has been no report regarding the association between the Planomicrobium okeanokoites and Himantothallus grandifolius; however, there are some reports on this bacterium isolated from sediments, soils, and lakes from Northern Hemisphere. This study may open a gateway for further research to know about the mode of interactions and how they affect the physiology and metabolism of each other.


Sample collection
The isolates of Himantothallus grandifolious collected from Larsemann Hills, Eastern Antarctica (69˚22ʹ24.6ʺS 76˚13ʹ41.9ʺE) during the 36 th Indian Scientific Expedition to Antarctica (ISEA) in 2016-2017. Samples were packed in sterile plastic zip-lock bags and stored at -80˚C for further studies. The representative specimen was pressed and deposited in the herbarium of Central University of Punjab, Ghudda, Bathinda, India, with voucher number WP-906: CUPVOUCHER-HiGr-2019-1.

Morphological examination of the algae
The samples were carefully washed in filtered sterile seawater (FSSW). Photographs were taken by using a bright-field microscope (BX53, Olympus, Japan), a digital SLR camera with Canon macro lens (EOS 60D, Japan). Analysis of the samples was done on the basis of their colour, texture, length of the thallus, shape, and arrangement of the cells. Public domain software ImageJ (http://rsbweb.nih.gov/ij/) was used for scale calibration and size measurements.

Isolation, cultivation, and biochemical characterization of the bacterium
The fronds were rinsed three times with filtered sterile seawater (FSSW) to remove loosely attached microbes and cut them into small pieces. The epiphytic bacterial strain was isolated through serial dilution in FSSW and plating techniques. The macroalgae surface was rubbed with a sterile cotton swab, and the extracted bacteria present in the cotton swab were inoculated in marine agar and marine broth 2216 (DIFCO). The inoculated plates were incubated at 10˚C and regularly inspected for growth for up to 4 weeks. Distinct colony morphotypes were restreaked on fresh medium until pure cultures were obtained based on their morphological characteristics by successive streaking using the ZoBell Marine Agar media (Himedia). The isolated bacteria were then observed for size, pigmentation, form, shape of the margins, and colony. The bacterium culture was used to observe the motility and Gram staining, which was confirmed through a low-power (10X) and high-power objective (40X) microscope. The bacterial strain was also examined for catalase, oxidase, and starch hydrolysis tests using standard protocols.

Molecular identification of isolates and phylogenetic analysis
DNA extraction, amplification, sequencing. Genomic DNA from epiphytic bacterial strain was extracted using HiPurA TM Bacterial genomic DNA extraction kit, and algal DNA was extracted using HiPurA TM Marine Algal DNA Purification Kit (HiMedia Laboratories Pvt. Ltd., Mumbai). The concentration of DNA was checked on a Nanodrop spectrophotometer. The 16S rRNA gene sequences were amplified by using the universal primers 27F (5'AGAGTT TGATCMTGGCTCAG-3') and 1492R (5'TACGGTTAACCTTGTTACGACTT-3') [32], The algal DNA were amplified by using universal rbcL (RuBisCO Large-subunit) [33,34], cox1 (cytochrome oxidase subunit1) [35], and LSU rRNA [36] primers sets with DreamTaq™ DNA Polymerase (Applied Biosystems, Foster City, CA, USA). PCR amplifications were performed in programmable thermal cyclers (Bio-Rad Laboratories) with initial denaturation at 95˚C for 3 minutes followed by 30 cycles of denaturation, annealing at 95˚C for 1 minute and 55˚C for 1 minute with final elongation step at 72˚C for 7 minutes [37]. The final PCR products were analyzed by 1.2% Agarose gel electrophoresis. The amplified sequences were purified using an Exo-SAP-IT1 PCR clean-up kit (USB Corporation, Cleveland, OH, USA) to avoid downstream interventions.
Purified PCR amplicons were subjected to bi-directional sequencing PCR using ABI Big-Dye Terminator Cycle Sequencing Ready1 Reaction Kit v3.1 (Applied Biosystems, Foster City, CA, USA). Sequencing reactions were purified using the traditional ethanol/EDTA precipitation method [38]. The dried samples were suspended again in 15 μl of Hi-Di™ Formamide and vortexed for 30 minutes, and then transferred to a sequencing plate for capillary gel electrophoresis (Applied Biosystems 3730 xl Genetic Analyzer, Foster City, CA, USA).
Sequence annotation and phylogenetic analysis. The sequence analysis and contig assembly were performed using licensed bioinformatics software Geneious1 prime v2020.0.4 (Biomatters Limited, New Zealand, available at https://www.geneious.com). The rbcL, COX1, and LSU sequences of algae and 16S rRNA sequences of bacteria were base call and annotated carefully. For sequence homology search BLASTn (www.blast.ncbi.nlm.nih.gov) was used. The newly generated sequences of Himantothallus grandifolius (accession no. MT274692, MZ676777, and MZ613320) and Planomicrobium okeanokoites (accession no. MT275689) were deposited in the NCBI Genbank database. Multiple sequence alignments were carried out using 16S rRNA gene sequences of the isolate, and other reference sequences were downloaded from the NCBI database (Table 3). These were aligned by the MUSCLE algorithm [39]. The end of aligned sequences was trimmed to reduce the number of missing sites across taxa and was aligned by MUSCLE algorithm in Geneious Prime.
Phylogenetic analysis of bacteria was conducted using Maximum Likelihood (ML) and Bayesian Inference methods. In Bayesian phylogenetic inference, MrBayes v3.2.6 plugin [40] was selected, followed by pairwise distance calculation. Pairwise distances between sequences of the samples were calculated using the nucleotide substitution test model in Geneious 1 Prime v2020.0.4. Bayesian analyses using the Markov chain Monte Carlo (MCMC) [41] technique was performed by MrBayes v3.2.6. The MCMC chains included four heated chains with 0.5 heated chain temperature and 200 subsampling frequencies. The Hasegawa-Kishino-Yano model [42] was used with a gamma-distributed variation 16S rRNA gene dataset and initiated an analysis from a random starting tree run for one million generations. The Maximum Likelihood method was performed using PhyML 3.3 in Geneious 1 Prime, and substitution bias was modelled by the Hasegawa-Kishino-Yano model with a gamma-distribution. A total of 1000 bootstrap replicates were examined under the ML criterion to estimate interior branch support. In the phylogenetic analysis, 17 nucleotide sequences were involved, and Bacillus subtilis ATCC 21331 (AB018487) was used as an out-group [43].

Results and discussion
According to the current understanding, the Antarctic macroalgae ecology appears to be hindered by the limited available database. In particular, a significant part of the Eastern Antarctic Coast between 45˚E and 160˚E is certainly under-sampled. Epiphytic bacteria that grow on the macroalgae surfaces live in a healthy competitive environment with limited space and access to nutrients. Many records are based solely on dredged or drift specimens, which are of limited usefulness or are doubtful because they were sampled only very few times and could have been confused with morphologically similar species. However, this is low species diversity relative to the world's temperate and tropical regions, but in a similar range as in the Arctic [44]. Heterokontophyta and Rhodophyta were found plenty in the marine environment. This research concerned isolating and detecting macroalgae-related epiphytic bacteria from Larsemann Hills, Eastern Antarctica. In this study, a strain of marine bacterium Planomicrobium okeanokoites associated with brown algae Himantothallus grandifolius was isolated. The present study made several fascinating revelations about the epiphytic and phylogenetic relationships between gram-positive bacteria and Antarctic macroalgae, as little information is available to date.

Morphological analysis of macroalgae
The thallus of the Himantothallus grandifolius appears dark-light brown, thick, and leathery, measuring around 45-50 cm in height, and is strap-shaped with ruffled margins (Fig 1A). The blades were tapered towards one end. Anatomically, the blade's anatomy shows separation across three layers. The outermost layer, meristoderm, is one cell thick, composed of rectangular cells deficient in physodes (Fig 1B). It is strap-shaped with ruffled margins (Fig 1A). The middle layer cortex consists of densely packed, sub-spherically shaped, parenchyma-like cells which contain physodes. The cells are very densely packed ( Fig 1C). The inner layer, the medulla, consists of plexus of rectangular cell filaments, which sometimes dichotomize ( Fig  1D). The appearance of sheathed trumpet hyphae is a characteristic feature of Himantothallus, which is also seen in running longitudinally ( Fig 1D).

Molecular analysis of macroalga
This study generated sequence information of Himantothallus grandifolius at Mitochondrial cytochrome c oxidase (cox1), chloroplast Ribulose-1,5 bisphosphate, and nuclear-Large ribosomal subunit (LSU rRNA) region. The sequences were submitted to GenBank, and the Accession numbers for rbcL, COX1, and LSU are MT274692, MZ676777, and MZ613320, respectively. NCBI top BLASTn hits for three loci are presented in Table 1. From this, we inferred that the specimen is Himantothallus grandifolius which belongs to the family Desmarestiaceae of Order Desmarestiales in Class Phaeophyceae.

Morphological and biochemical characterization of an isolated bacterium
The isolated epiphytic bacterial strain with colonies bearing pale yellow to orange in colour (Fig 2) and rod-shaped motile cells. The biochemical assay showed a positive response for catalase, urease, and oxidase and negative for the starch hydrolysis test. The comparative results of biochemical determinations with different species of Planomicrobium with new epiphytic bacterial strain showed in Table 2.

Phylogenetic analysis of epiphytic bacterium
Phylogeny of the 16S rRNA sequence with additional 16 nucleotide sequences were involved in constructing a phylogenetic tree where Bacillus subtilis ATCC 21331 (AB018487) was used as an out-group. For phylogenetic analysis, sequences were first aligned by the MUSCLE algorithm in MEGA. In clade one, the isolate clustered with other P. okeanokoites strains of related taxa procured from the NCBI database with 1.00 PP/97% bootstrap values. Different reports on Planomicrobium sp. with their isolation sources/hosts are given in Table 3.
The NCBI BLASTn searches of 16S rRNA gene sequences of isolated epiphytic bacterial strain showed the best matches (based on percentage identity) >98.78% with Planomicrobium

PLOS ONE
First report of Planomicrobium okeanokoites from Southern Hemisphere okeanokoites SLA-357 (MT125787). Top ten BLASTn hits of the bacterial sequence showed approx. 98% similarity to the isolated epiphytic bacterial sequence (Table 4). 16S rRNA gene sequence of epiphytic bacterium generated in this study was 1394 base pair (bp) in length. A phylogenetic tree based on 16S rRNA gene sequences was constructed using the maximumlikelihood (ML) and Bayesian inference methods (Fig 3). Values at the nodes indicate posterior probability support and bootstrap values. Full statistical posterior probability support (1.00 PP/100% PP). The highly supported branches are shown in bold (Posterior Probability > 0.95 calculated with MrBayes and bootstrap values >95 using maximum likelihood). From morpho-phylogenetic studies, it can be concluded that the isolated bacterium is Planomicrobium okeanokoites which provides new insights into understanding the epiphytic associations between bacterial strains and the algal microbiome in Antarctica. Macroalgae provide nutrients and shelter to bacteria as a by-product of their photosynthesis, whereas bacteria

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First report of Planomicrobium okeanokoites from Southern Hemisphere provide vitamins, such as Vit B 12 [45] and growth factors [46] for algal growth [47,48]. Such intimate epiphytic associations proved macroalgae and bacteria as a holobiont or unified functional entity. The analysed data have been validated with the isolated bacterium Halimeda sp. associated with green algae from Lake Kakaban, Indonesia [49]. This research was also validated by three Antarctic subtidal macroalgae (Himantothallus grandifolius, Pantoneura plocamioides, and Plocamium cartilagineum) by a similar study, two of them were investigated as a source for isolation of agar-degrading bacteria, identified based on 16S rRNA belonged to the genera Cellulophaga, Colwellia, Lacinutrix, Olleya, Paraglaciecola, Pseudoalteromonas and Winogradskyella [50].

Conclusion and prospectives
This study gives a first report on the presence of a gram-positive bacterium Planomicrobium okeanokoites, from the surface of Himantothallus grandifolius (Desmarestiales, Phaeophyta), from Larsemann Hills, Eastern Antarctica. In addition, this is the first report of  Planomicrobium okeanokoites from the Southern Hemisphere to the best of our knowledge. This study is based on the chemotaxonomic and morpho-phylogenetic identification of Planomicrobium okeanokoites from Himantothallus grandifolius. However, there are some reports on this bacterium from sediments, soils, and lakes from the Northern Hemisphere. This study has a taxonomic importance and provides insights into understanding the origin of life of the bacterium in a very harsh climate conditions. This species might be introduced by some means or may be already existing there. Some studies have described the role of an epiphytic bacterium in influencing the metabolism and morphology of the host plant [51,52]. For

PLOS ONE
First report of Planomicrobium okeanokoites from Southern Hemisphere example, a bacterium associated with Ulva mutabilis is responsible for the development of blade morphology, its adhesiveness to the substratum, and growth [53]. This study may open a gateway for further research to know about the mode of interactions and how they affect the physiology and metabolism of each other. This study sets a prospect to understand how bacterial strains are associated with the algal microbiome in Antarctica and the critical processes involved in the association. However, this study revealed the identification of the bacterium Planomicrobium okeanokoites from Larsemann Hills, Eastern, Antarctica on the Himantothallus grandifolius (Desmarestiales, Phaeophyta). However, future research is needed to infer the compounds involved in epiphytic association of the bacterium Planomicrobium okeanokoites with Himantothallus grandifolius.